A regulatory mechanism governing glycolysis in brain has been found as a result of the observation that glucose 6-phosphate (G6-P) accumulates in an unsupplemented supernatant of rat brain and the accumulation can be enhanced by the addition of phospholipid. A study of the fates of the hexose phosphates in the presence and absence of phospholipid shows that phospholipid affects the irreversible interconversion of the fructose phosphates, causing G6-P to accumulate in the wake of inhibition of glycolysis by either stimulation of fructose 1,6-bis-phosphatase or inhibition of phosphofructokinase or both. The source of G6-P is UDPglucose, itself derived from an endogenous precursor.